Post on 05-Jan-2017
Major Cellular and Physiological Impacts of OceanAcidification on a Reef Building CoralPaulina Kaniewska1*¤, Paul R. Campbell2, David I. Kline1, Mauricio Rodriguez-Lanetty3, David J. Miller4,
Sophie Dove1,4., Ove Hoegh-Guldberg4,5.
1 School of Biological Sciences, The University of Queensland, St Lucia, Queensland, Australia, 2 Agri-Science Queensland, Department of Employment, Economic
Development and Innovation, Dutton Park, Queensland, Australia, 3 Department of Biology, University of Louisiana at Lafayette, Lafayette, Louisiana, United States of
America, 4 ARC Centre of Excellence for Coral Reef Studies and Coral Genomics Group, School of Pharmacy and Molecular Sciences, James Cook University, Townsville,
Queensland, Australia, 5 Global Change Institute, The University of Queensland, St Lucia, Queensland, Australia
Abstract
As atmospheric levels of CO2 increase, reef-building corals are under greater stress from both increased sea surfacetemperatures and declining sea water pH. To date, most studies have focused on either coral bleaching due to warmingoceans or declining calcification due to decreasing oceanic carbonate ion concentrations. Here, through the use ofphysiology measurements and cDNA microarrays, we show that changes in pH and ocean chemistry consistent with twoscenarios put forward by the Intergovernmental Panel on Climate Change (IPCC) drive major changes in gene expression,respiration, photosynthesis and symbiosis of the coral, Acropora millepora, before affects on biomineralisation are apparentat the phenotype level. Under high CO2 conditions corals at the phenotype level lost over half their Symbiodiniumpopulations, and had a decrease in both photosynthesis and respiration. Changes in gene expression were consistent withmetabolic suppression, an increase in oxidative stress, apoptosis and symbiont loss. Other expression patterns demonstrateupregulation of membrane transporters, as well as the regulation of genes involved in membrane cytoskeletal interactionsand cytoskeletal remodeling. These widespread changes in gene expression emphasize the need to expand future studiesof ocean acidification to include a wider spectrum of cellular processes, many of which may occur before impacts oncalcification.
Citation: Kaniewska P, Campbell PR, Kline DI, Rodriguez-Lanetty M, Miller DJ, et al. (2012) Major Cellular and Physiological Impacts of Ocean Acidification on aReef Building Coral. PLoS ONE 7(4): e34659. doi:10.1371/journal.pone.0034659
Editor: Wei-Chun Chin, University of California, Merced, United States of America
Received September 19, 2011; Accepted March 6, 2012; Published April 11, 2012
Copyright: � 2012 Kaniewska et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Australian Research Council Centre for Excellence for Coral Reef Studies. The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: p.kaniewska@aims.gov.au
. These authors contributed equally to this work.
¤ Current address: Australian Institute of Marine Science, Townsville MC, Queensland, Australia
Introduction
Coral reefs are highly productive and biologically diverse
ecosystems despite the oligotrophic waters that surround them [1].
They are important to millions of coastal dwelling people across
the planet, underpinning industries such as fishing and tourism [2].
Coral reefs appear to be facing a significant increase in local and
global stressors [1,3]. Global warming and ocean acidification
have recently emerged as key threats to the long-term survival of
coral reefs. Rapidly warming oceans are driving an increase in the
frequency and intensity of mass bleaching events [3], while steadily
acidifying oceans have caused large decreases in the concentration
of carbonate ions and potentially the ability of marine calcifiers to
precipitate calcium carbonate [4].
High levels of atmospheric CO2 ([CO2]atm) and subsequent
ocean acidification have been implied as a major factor in several
extinction events on coral reefs in geological time [5]. The ocean
uptake of [CO2]atm produces carbonic acid (HCO32) as the
carbon dioxide reacts with water. Protons (H+), which are formed
due to the resulting dissociation of carbonic acid to bicarbonate
ions (CO322), react with carbonate ions, forming more HCO3
2
and thus reducing carbonate ions available for marine organisms
[6]. This decrease in [CO322] leads to a reduction in the
saturation state of calcium carbonate forms such as aragonite,
calcite and high magnesium calcite and thus a reduction in
calcification by marine organisms [4,7]. To date, most studies of
ocean acidification have focused on its impact on calcification
rates [4], as opposed to targeting the physiological processes that
lead to the biological deposition of calcium carbonate in these
organisms and/or sustain organism health (fitness). It is now clear
that overall the predicted reduction in ocean pH and [CO322] can
be correlated with a decrease in calcification for a diverse range of
marine calcifiers, however the response is variable, often non
linear and there are inter and intra specific differences [4,8,9]. In
addition, for studies conducted in the field, ocean acidification
effects can be compounded by ocean warming [10]. Calcification
is clearly important, but many other physiological processes may
be affected in marine organisms [11,12,13]. By assessing these
impacts we can commence unraveling cellular and physiological
processes that eventually lead to a decrease in calcification rates.
This in turn can provide information to explain currently observed
discrepancies in calcification rates, which is important if we are to
PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e34659
understand the full ramifications of rapid ocean acidification for
coral reefs. Here, we investigate what physiological processes in
Acropora millepora are affected by changes in ocean pH, both at the
level of the phenotype and gene expression level and show that
exposure to high CO2 drive major changes in gene expression,
respiration, photosynthesis and symbiosis for the reef building
coral.
Results and Discussion
In a study of 8606 unigenes from the coral Acropora millepora
exposed to ambient, mid and high CO2 conditions as predicted by
the IPCC (Table 1), we report that increases in dissolved CO2 after
1 and 28 days affected processes including: metabolism, mem-
brane-cytoskeleton interactions, signaling, translation, transport,
calcification, protein folding and apoptosis (Figure 1, Table S1). In
total, acidification resulted in 643 differentially expressed tran-
scripts (FDR, 5%); the largest number of these differentially
expressed genes are up or down regulated in the high CO2
treatment compared to the control at day 28. This was also
reflected in principal component analysis which showed that high
CO2 corals at day 28 where separated from the other samples
implying the greatest variation (Figure S1). Differentially expressed
genes were subjected to K-means clustering in order to group
genes with similar temporal expression patterns and we identified
6 major synexpression clusters (I–VI) (Figure 1). Transcripts with
homology to known genes (352 transcripts, Blastx, E-score cutoff
1026) were assigned to gene ontology (GO) categories and
subjected to classification analysis to identify enriched GO groups
(Figure 2). From the pie charts in Figure 1 which show what major
GO categories genes in the synexpression clusters belong to, it is
apparent that more changes in cytoskeleton interactions occur in
cluster IV, more changes in signaling and catalysis occur in clusters
I–III and large changes in transport occur in cluster II.
Quantitative real-time PCR of ten representative genes supported
the results, where each candidate gene in the qPCR followed the
trends found in the microarray data with expression levels either
increasing or decreasing in response to high CO2 conditions
(Figure 3, Table S2, S3) compared to control corals at day 28.
Changes in response to high and mid CO2 conditions for day 1,
where less gene expression changes occurred, contained many
changes in heat shock proteins and signaling which differed from
changes at day 28 (Table S1). However, this study only had a
single time point at a shorter time scale. It would be useful in
future studies to better define changes in gene expression levels
within the first few days of exposure, which would require an
experiment with several time points within these first few days. It
should be noted that this study used small sample sizes (n = 3 for
microarray analysis and n = 4 for physiology and qPCR) and that
future studies would benefit from greater sample sizes, perhaps a
greater range of differentially expressed genes would be detected,
and more robust conclusions drawn from the physiological data.
Changes at the mRNA level, where the majority of differentially
expressed genes were found at day 28 in the high CO2 treatment,
were confirmed by physiological measurements (Fig. 4). Acropora
millepora branches lost Symbiodinium cells in response to changes in
ocean chemistry, (Figure 4A; Kruskal Wallis test, H
2,12 = 7.54,p = 0.023), where after a 28 day exposure, Symbiodinium
populations in the high CO2 treatment were reduced
(1.02610665.346104) to less than half the density compared to
control branches (2.3610664.686105) (Figure 4A). The remain-
ing symbiont cells also became less productive and the photosyn-
thetic capacity (as measured by Pnet max cell21 and Pgross max
cell21) was reduced. There was a 60% reduction (Kruskal Wallis
test, H 2,12 = 8.34, p = 0.015) in Pnet max cell21 and a 50%
reduction (Kruskal Wallis test, H 2,12 = 7.73, p = 0.021) in Pgross
max cell21 (Pnet max2LEDR) in the high CO2 treatment
compared to the control (Figure 4B). Decreasing rates of gross
photosynthesis per Symbiodinium cell, compounded by reduced
Symbiodinium populations, may lead to a reduction in photoassimi-
lates translocated to the host coral. These changes are likely to
have long-term negative effects on host growth and fecundity, with
the prospect of increased susceptibility to disease and mortality,
especially if Symbiodinium populations fail to recover rapidly [14].
The observed decrease in Pgross max is consistent with previous
acidification studies [11,15]; however, in our study there was no
change in LEDR per remnant Symbiodinium cell among CO2
conditions (Kruskal Wallis test, H 2,12 = 1.65, p = 0.437). This may
be due to the application of very different light conditions to
Crawley et al [15] which exposed coral to sub-saturation light
intensities and only had a short experimental time scale. More
importantly, there was a 3-fold downturn in dark respiration per
coral surface area (Figure 4C), (Kruskal-Wallis test, H 2, 12 = 6.71,
p = 0.035), which is typically associated with a decline in host
maintenance and/or growth [16]. Rapid growth, either as tissue
growth or calcium carbonate deposition necessitates high respira-
tion rates, but the observed reductions in the rate of respiration
can suggest suppression of growth rates and/or metabolism.
Physiological changes in this study preceded any observable
changes in calcification/growth as determined by changes in
buoyant weight, as there was no difference in branch calcification/
growth rates between the 3 treatments after the 28 day incubation
(Kruskal Wallis test, H 2,12 = 0.50, p = 0.778) (Figure 4D), despite
the downturn in both energy production and respiration observed
in the high CO2 treatment. This result may reflect that in this case,
observable effects on calcification/growth rates require longer
experimental incubation, as the buoyant weight technique may be
too insensitive to measure the potential small changes in
calcification/growth that may have occurred.
Table 1. Carbonate chemistry parametersa across experimental conditions.
IPCC pH* ALK* (mM/kgSW) DIC* (mM/kgSW) (Aragonite) pCO2 (matm) CO322 (mmol kg21)
Control (present) 8.0–8.2 2281.9615.8 1832.4659.4 3.93–5.21 260–440 253.8617.9
A1B (medium) 7.8–7.9 2260.0612.6 2165.4651.0 1.14–3.71 600–790 145.3633.7
A1FI (high) 7.6–7.7 2283.3613.5 2345.56214.4 0.77–2.85 1010–1350 89.5613.0
*Measured values.aParameters were calculated from measured values of pH, total alkalinity (ALK), dissolved inorganic carbon (DIC), temperature (256C) and salinity(35 ppm), using the program CO2SYS.doi:10.1371/journal.pone.0034659.t001
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MetabolismChanges to metabolic rates are a common outcome of
environmental stress [17]. Changes in gene expression suggest
that Acropora millepora may have reduced its metabolism under high
CO2 conditions at day 28 (Figure 1 cluster IV–VI, Figure 2, Table
S1), mirroring the oxygen flux change (Figure 4). There was an
overall down-regulation of genes involved in the tricarboxylic acid
(TCA) cycle and the mitochondrial electron transport chain (Table
Figure 1. Graphical representation of differentially expressed genes in Acropora millepora across pCO2 treatments (control, mediumand high) at day 1 and 28. K-means clustering was applied to group genes (synexpression clusters I–VI) by common temporal expression patterns.Yellow represents upregulation and blue represents downregulation, scale bar is on a log2 ratio. Each row corresponds to a transcript and eachcolumn represents the mean expression (n = 3). For each cluster average log2 fold changes (6SE) at day 28 are indicated and pie charts classify genesinto major biological processes according to enriched GO categories.doi:10.1371/journal.pone.0034659.g001
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S1), indicating reduced oxidative metabolism and capacity to
generate ATP and NADPH. There was also an upregulation of
triglyceride lipase and Acyl-CoA dehydrogenase (Figure 1 cluster I,
II, Table S1), which may point to an increase in the breakdown of
lipids for energy use [18,19]. Interestingly, there was an increase in
mitochondrial transcripts for ATPase (Figure 1 cluster II, Table S1).
Cellular apoptosis is often preceded by an increase in mitochondrial
ATPase activity, resulting in an influx of potassium, the activation of
caspases and ultimately cell death [20]. Metabolic suppression has
been shown in a range of marine organisms in response to CO2
fluctuations [13,21]. The majority of energy needs in tropical reef
building corals are supplied by the photosynthetic endosymbionts
[22], but host heterotrophy can occasionally meet host requirements
[23]. Depressions in aerobic metabolic activity due to mitochondrial
disruptions can undermine the viability of host cells regardless of the
trophic source of organic carbon supplied into the TCA cycle. In
this particular case, metabolic suppression due to acidosis is likely to
have long-term fitness costs.
Acid-Base Regulation and Ion/Macromolecule TransportMaintaining pH homeostasis is critical to a range of cellular
functions [24]. Studies of acid-base regulation and hypercapnia
suggest significant physiological challenges for marine fish and
worms [13,25]. There are cases where mitochondrial energy
production is tied to acid-base regulation through HCO32
transport [26], bi-direction H+ pumping by F0F1 ATPase [20],
or Na+/H+ and Cl2/HCO32 transporters on the cell membrane
[25]. Membrane proteins play an integral role in: pH homeostasis
of the cell, membrane lipid composition and cell shape
maintenance [27]. For A. millepora, 28 days of high CO2 conditions
resulted in changes in membrane transporters (Figure 1, 2, Table
S1). Specifically, there was downregulation of proton channels (V-
type proton ATPases), phosphate transport and protein transport
at the cell membrane (Figure 1 cluster IV,V, Table S1). At the
same time, sodium and potassium transporters, cell membrane
receptors and an ABC transporter were upregulated (Figure 1
cluster I, Table S1). In eukaryotes, ABC type transporters are
involved in the export of unwanted molecules, such as toxins [28]
from the cell. V- type proton ATPases at the cell membrane serve
to acidify the extracellular environment which in turn activates a
series of signaling cascades [29]. In the cnidarian ectoderm,
plasma membrane proton ATPase activity has been tied to CO2
uptake [30]. A decrease in this transporter may indicate a decrease
in CO2 uptake under acidification stress. Due to concurrent
increases in energy saving ion gradient transporters such as Na+/
H+ exchangers, the decrease in V- type ATPases for proton
transport may also be the result of an active suppression of the
more costly ATP dependent ion transporters [31]. In addition, a
lipid transporter was upregulated in the high CO2 treated corals at
day 28 (Figure 1 cluster II, Table S1), a change not found in acid
base regulation of other marine organisms [13,21,25], perhaps
implying changes to the lipid configuration of the cell membrane
as a response to ocean acidification [32].
Stress Response MechanismsAbiotic changes are likely to elicit a cellular stress response
(CSR), a universally conserved mechanism to protect macromol-
ecules within cells from the potential damage that physical,
chemical or biological stressors may cause. The CSR can increase
the tolerance temporarily to the stressor, and remove already
damaged cells through apoptosis [33]. Transcripts of A. millepora
that encode a number of cellular defenses, and transcripts involved
Figure 2. Classification analysis for Acropora millepora transcripts that were differentially expressed across pCO2 treatments(control, medium and high) at day 1 and 28. Gene enrichments (P,0.05) across GO categories are shown. The program GOEAST was used totest for enriched GO categories among differentially expressed genes. Color scheme indicates parent categories (binding, actin cytoskeleton, catalyticactivity, metabolic processes and transporter activity) and individual pie segments are annotated for more specific GO categories. The sizes of the piesegments are proportional to the total number of genes enriched. The proportion of differentially expressed genes which were assigned to geneontology categories was 55%.doi:10.1371/journal.pone.0034659.g002
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in maintenance of protein integrity (molecular chaperones) were
downregulated (Figure 1 cluster V, Table S1), whilst genes,
protecting the cells against oxidative stress through oxidoreductase
activity (eg. Catalase, FAD linked oxidase and selenoprotein
[34,35,36]) and involved in apoptosis (caspase 3, TRAF3, p53
inducible protein 11 and programmed cell death protein 4
[37,38,39]), were upregulated in high CO2 treated corals at day 28
(Figure 1 cluster I, II, III, Table S1). Bcl-2, MALT1 and API-5,
potential inhibitors of apoptosis [40,41,42] were downregulated
(Figure 1 cluster V, Table S1). The upregulation of apoptotic
transcripts is consistent with the upregulation of mitochondrial
ATPase described above, which together point to disruption in the
mitochondrion leading to cell death [20]. An increase in apoptosis
may reflect that prolonged environmental stress, and either a lack
of cell pH homeostasis or elevated maintenance costs, has resulted
in cell damage. The loss of Symbiodinium cells and an increase in
transcripts alleviating oxidative stress may point to impairment in
the photosynthetic apparatus in the dinoflagellate symbiont or an
impairment of the coral mitochondria [14]. This, in turn, would
increase the presence of oxygen radicals in the host tissues and
imply cell damage potential. The fact that high CO2 conditions
resulted in overall downregulation of protein folding transcripts,
may be a sign that the coral tissue may no longer have the capacity
to maintain these integral services. Interestingly, at day one of the
high CO2 treatment there was an upregulation of Heat shock
protein 40, a change not found at Day 28 (Figure 1 cluster II,
Figure 3. Log2 relative expression of selected genes using quantitative real-time PCR. Expression levels of genes are plotted as ratio ofrelative expression of high CO2 corals versus control corals at day 28. The relative expression for these selected genes was normalized to AdoHcyaseand Rbl7. Bars represent standard error of the mean (n = 4).doi:10.1371/journal.pone.0034659.g003
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Table S1). It must be noted however, that the two other main heat
shock proteins (hsp) were not differentially expressed between
treatments (hsp 70 and 90) but were maintained at a high
expression levels, and their presence may be sufficient for the
integrity of newly made proteins. Calnexin and alpha mannosidase
transcripts were upregulated (Figure 1 cluster II, Table S1) which
would increase the quality control and protein folding ability in the
endoplastic reticulum for proteins that will then be further
transported to the golgi complex [43,44]. It is possible that the
other downregulated chaperones could be temporarily reduced
while awaiting more favorable environmental conditions. A coral
c-type lectin, which is involved in innate immunity in corals [45]
was downregulated (Figure 1 cluster V, Table S1) under high CO2
conditions, indicating that this cell stress response may not be
responding appropriately, and this decrease may compromise the
coral further as in a stressed holobiont, susceptibility to pathogens
may increase [46].
Ca2+ Ion Binding/Transport and Cell CommunicationSeveral calcium (Ca+) ion binding proteins were downregulated
in high CO2 treatments at day 28 (Figure 1 cluster IV, V, Table
S1). Transcripts for calcium-binding receptors that are potentially
involved in innate immunity [47,48] were also suppressed,
implying an adverse change in signaling potential at the cell
membranes. Downregulation of calmodulin, FKBP12 and EGF-
hand proteins also implies potential disruption in cell calcium
homeostasis [49,50,51], as these calcium binding proteins control
the Ca2+ release from ryanodine receptors (RyR) within the
endoplasmic reticulum (ER), which is an intracellular Ca2+ storing
organelle [49,50,51,52]. Calpain, an important Ca2+ activated
protease that has roles in membrane-cytoskeleton interactions,
signal transduction, cell differentiation and apoptosis [53], was
upregulated. Changes in these calcium binding proteins indicate
that certain signaling pathways may have been altered.
Membrane-Cytoskeleton InteractionsExposure to high seawater CO2 concentrations for 28 days
resulted in several differentially expressed genes involved in
membrane-cytoskeleton interactions and cytoskeletal remodeling
(Figure 1, 2, Table S1). It is possible that the change in regulation
of these transcripts reflects a change in proteins involved in
cytoskeletal interactions, cytoskeletal organization, intracellular
Figure 4. The effect of increasing CO2 in seawater (control, medium and high) after 28 days on coral-algal physiology. (A)Symbiodinium cell number in reef-building coral, Acropora millepora (B) photosynthetic capacity per symbiont cell measured as Pnet max, lightenhanced dark respiration (LEDR), and Pgross max (Pnet max2LEDR) (C) dark respiration(R dark) and (D) relative calcification/growth as % change inweight (g) of coral branches over the 28 day experimental period. Error bars represent the standard error of the mean (n = 4).doi:10.1371/journal.pone.0034659.g004
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transport, cell shape integrity and cell motility [54,55] [55].
Specifically, there was downregulation of cytoskeletal actin 1,
centractin, radixin and coatomer epsilon subunit and radixin
(Figure 1 cluster IV, V, Table S1), whilst there was upregulation of
tubilin and Lgl tumor suppressor unit (Figure 1 cluster I, II, Table
S1). The actin cytoskeleton is important in a diverse range of
processes such as cell motility, contractibility, mitosis and
cytokinesis, intracellular transport, endocytosis and secretion. In
addition, it has been suggested that actin is also involved in
regulation of gene transcription through changes in the cytoskel-
etal actin dynamics or assembly of transcriptional regulatory
complexes [55]. Actin is also an important part of the nuclear
complex being required for the transcription of RNA polymerases
and is also involved in the export of RNAs and proteins from the
nucleus [55]. It is possible that the downregulation of cytoskeletal
actin in high CO2 conditions reflects a change in the regulation of
gene transcription of proteins involved in cytoskeletal interactions.
In addition this downregulation can imply changes in the
intracellular transport, plasma membrane interactions and cell
shape/integrity. There was also an upregulation of alpha tubilin,
which forms a constituent of the microtubule filaments, involved in
cytoskeletal organization and vesicle transport. Downregulation of
coatomer epsilon subunit implies changes in protein trafficking
between the endoplasmic reticulum and the Golgi complex, while
upregulation of Lgl tumor suppressor unit indicates changes to
events controlling cell polarity [56,57]. Cell volume control
changes have been recorded in other marine organisms such as
crabs in response to hypercapnia [58], and similar changes may be
occurring in the stressed coral cells. The downregulation of
Radixin, an important protein involved in linking the plasma
membrane to the cytoskeleton using actin rich surfaces [59],
supports the downregulation of cytoskeletal actin. Centractin, or
Actin Related Protein 1 (ARP1), was also downregulated under
higher CO2 stress, and this is an important activator of
cytoplasmic vesicle movement [60]. In contrast, at day one in
high CO2 stressed corals, there was an upregulation of Radixin
and Centractin, or Actin Related Protein 1 (ARP1) (Figure 1
cluster VI, Table S1), indicating that different changes in
cytoskeletal interactions were occurring at this stage. The
cytoskeleton has profound effects on the plasma membrane. At
times, there may be uninhibited lateral diffusion of lipids and
proteins across the plasma membrane; the influx of these
molecules can be regulated by the membrane-cytoskeleton links.
These become obstacles to free diffusion through diffusion-limited
lipid domains [54]. It may be that changes in these membrane-
cytoskeleton links in this study reflect changes in transport across
the membrane.
Rab/Ras GTPasesExposure to increased CO2 concentrations for 28 days lead to
an up (Figure 1 cluster II, Table S1) and downregulation (Figure 1
cluster V, Table S1) of transcripts that resemble members of the
Rab/Ras GTPases families (small G-proteins). The Ras GTPase
superfamily is a small monomeric group of GTPases, which are
involved in cell proliferation and cell signaling events in response
to external stimuli. Disruption of the Ras signaling pathway is a
key component in the progression of tumor growth [61]. The Rab
GTPase family is part of the Ras GTPase superfamily and plays a
key role in many membrane-trafficking events in eukaryotic cells,
such as exocytosis. This group of proteins which tightly associates
with the cell membrane is involved in transport vesicle formation,
actin and tubilin motility, docking and membrane fusion. Rab
proteins are active when bound to GTP and are inactive when
bound to GDP [62,63]. In its active state, the Rab protein
regulates the transport of lipids and proteins between distinct
membrane bound organelles through interactions with down-
stream effector proteins which are recruited onto the membranes
[63]. Small G-proteins are implicated in most cellular events
where plasma membrane-cytoskeleton interactions or plasma
membrane shape changes (plasma membrane deformations)
occur. The observed upregulation in members of these small
monmeric GTPases most likely reflects changes in the cell
membrane and cytoskeletal interactions to accommodate changes
in external seawater chemistry.
Extracellular matrixChanges in the extracellular matrix (ECM) have previously
been implied to potentially affect calcification [34,36]. Our
expression patterns indicate that only two transcripts encoding
previously described ECM proteins changed after 28 days under
mid CO2 exposure. SEC13L1 was upregulated (Figure 1 cluster
III, Table S1) while peroxidasin was downregulated (Figure 1
cluster IV, Table S1). At day 28 under high CO2 exposure, there
was also a downregulation to a predicted protein in the
extracellular matrix (Table S1). This implies that small changes
to calcification may have started occurring and that perhaps with a
longer experimental incubation time more ECM and calcification
related transcripts would have been differentially expressed. This
overall supports our findings at the phenotype level where no
change in calcification/growth was found (Figure 4). Overall in
this study there were fewer changes in transcripts which may be
involved in calcification, in response to ocean acidification,
compared to gene expression studies with corals exposed to
thermal stress where changes to the following transcripts were
observed; collagen a-1, ECM matrix metalloprotease, papilin,
carboxypetidase inhibitor SmC1, procollagen, galaxin and SCP-
like extracellular protein [34,36].
Cell-wide Responses by Corals to Ocean Acidification: AModel
To highlight the differences between acidosis which may be a
factor in this study from the impact of hypercapnia seen in other
marine organisms, we purpose a model (Figure 5) of cell-wide,
coral host response to high CO2 stress. This model attempts to
account for the classic acidosis response (acid-base regulation and
metabolic depression) and the novel responses observed in the
studied coral (apoptosis, signaling events, calcium homeostasis,
cytoskeletal remodeling, cytoskeletal-membrane interactions and
oxidative stress). The coral specific responses may result from
increased reactive oxygen species (ROS) and/or increased reactive
nitrogen species (RNS) created from a disturbance in the
Symbiodinium cell, the host mitochondria, or both [14,34].
Upregulation of catalase, FAD-linked oxidase and selenoprotein
indicates that there may be an increased amount of ROS in the
coral cells [64,65]. Increased ROS/RNS can result in a disruption
to the calcium homeostasis [34]. The role of internal [Ca2+]
increase in coral bleaching has been suggested previously [34].
The downregulation of calmodulin (CaM), FKBP12 and EF-hand
proteins under high CO2 stress indicates that there may be a
disruption to the Ca2+ homeostasis [49,50,51]. Modifications of
the actin cytoskeleton, membrane-cytoskeleton interactions and
cell receptor/adhesion properties will be affected by a disruption
in Ca2+ homeostasis and metabolic suppression [34,66]. Both
oxidative stress and an increase in intracellular Ca2+ can lead to
apoptosis and changes in transcripts indicate that both the NF-kB
and p53 apoptotic pathways [67,68] were upregulated. Changes in
predicted proteins in the extracellular matrix may imply changes
in or restructuring of the extracellular matrix. Our model suggests
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that similar cellular events are occurring under acidosis induced
bleaching to those reported for thermally induced bleaching
[14,34,36], but with the addition of changes to acid-base
regulation and mitochondrial ATPase activity.
On our present greenhouse trajectory, we are likely to use all the
.4000 Gt of carbon present in the global fossil fuel reserves by
2400. This will significantly acidify the oceans for thousands of
years [69] and take them to a point not seen in tens of millions of
years [70]. Our study highlights the imperative to investigate the
impacts of ocean acidification on processes other than those
involved in biomineralisation. Also, there is a need for more
studies investigating the effects of naturally occurring changes in
pCO2 on marine calcifiers in situ. This is a priority, if we are to
understand the fate of the many supporting roles that corals
contribute to the maintenance of coral reefs.
Materials and Methods
Experimental DesignA total of 20 branches (7–8 cm long) were collected from 4
healthy colonies of the reef building coral Acropora millepora on
Heron Island Reef flat (23 339S, 151 549E), Great Barrier Reef,
Australia. Coral branches were affixed onto cut 15 mL falcon
tubes using Selleys Knead It Aqua (Padstow, Australia) and Selleys
autofix super glue (Padstow, Australia). The affixed branches were
then placed onto a rack which was deployed back to the Heron
Figure 5. A proposed model of cellular events occurring as a result of ocean acidification. These changes lead to compromised health inAcropora millepora (reduction in symbiont cells and decreased photosynthesis and respiration). The schematic depicts an endodermal cell whichcontains the symbiont cell. Cellular events depicted here are likely to also occur in other cell types which do not contain symbionts, especially theacid base changes at the cell membrane. Changes in carbonate chemistry lead to changes in acid base regulation and cell membrane transporters.Acid base regulation may not be sufficient leading to acidosis within the cell. This could increase reactive oxygen species (ROS) due to a disruption(q) in the Symbiodinium cell (S) and/or in the coral host mitochondrion (M), which may also produce reactive nitrogen species (RNS). The overalloxidative stress and a disruption to calcium stores at the endoplasmic reticulum (ER) can lead to calcium imbalance. This in turn leads to events suchas changes in the extracellular matrix, cytoskeletal remodeling, changes in cytoskeletal interactions, disruption to cell reception and signalingpotential, and an increase in cell death. Moreover disruption in both the host mitochondrion and Symbiodinium cell leads to a decrease inmetabolism and a decrease in metabolite transfer from the symbiont cell. In addition the disruption in the host mitochondrion can also lead to celldeath. For the cell membrane transporters black arrows indicate upregulation and white arrows indicate downregulation.doi:10.1371/journal.pone.0034659.g005
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PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e34659
Island reef flat, where they remained for 4 weeks, exposed to
natural light and flow regimes in order to recover from handling.
Following this acclimation period they were transferred to aquaria
with running seawater and under ambient light (with shade cloth
see below) and ambient temperature (26uC) conditions for 10 days.
For each treatment there were four randomly distributed aquaria
and for each A. millepora colony, branches were evenly distributed
across treatments, with 6 branches per colony in each of the
treatment tanks (ambient, mid and high – see below). Branches
were designated to be used either for respirometry assays and
physiology measurements or microarray analysis. The experiment
was run for 28 days and coral branches were sampled, snap frozen
in liquid nitrogen and stored at 280uC for later analysis at time
zero, day 1 and day 28. For each time point, two branches per
colony were sampled and one branch was used for physiology and
one branch was used for genomic analysis.
The experimental set up consisted of 12 (4 aquaria per
treatment) flow-through aquaria (80 L) under natural light and a
layer of shade cloth which resulted in photosynthetically active
radiation levels of a maximum of 859.6 mmole quanta m22 s21
and a daily average level of 433.668.6 mmole quanta m22 s21 for
the light period of the day. Aquaria were supplied with unfiltered
seawater which was being pumped straight from the reef flat on
Heron Island into CO2 mixing tanks, and then distributed across
aquaria. The control aquaria were receiving seawater from the
Heron Island reef flat where the natural diurnal variability in pH
ranged between 8.0–8.2 due to tidal changes and metabolic
activity on the reef flat, which corresponded to a pCO2 range of
260–440 ppm. The pCO2 ranges in the two acidification
treatments were controlled by a CO2 dosing control system
(Aquacontroller III, Neptune Systems, Carlsbad, CA, USA) which
used pH readings in the large 300 L CO2 mixing tanks, to either
open or close solenoid valves (Dupla Australia, Littlehampton,
Australia) and would control the amount of CO2 being added to
the mixing tanks. The medium CO2 treatment was controlled to a
pH target of 7.8–7.9 corresponding to 600–790 ppm. The high
CO2 treatment was targeted to a pH range of 7.6–7.7
corresponding to 1010–1350 ppm. Temperature, pH and light
levels were recorded throughout the experiment and total
alkalinity across control and CO2 treated aquaria was determined
with a Mettler Toledo T50 automated titrator, with 0.1 M HCl
and 130 g seawater samples using the Gran titration method in a
two-stage, potentiometric, open-cell titration following the method
of [71]. Acid concentrations and the alkalinity measurements were
calibrated at the beginning of each run using Dickson certified
reference sea water standards (Andrew Dickson, SIO, Oceanic
Carbon Dioxide Quality Control). Dissolved Inorganic Carbon
(DIC) was sampled into 50 mL glass vials after filtering with a
0.45 mM syringe filter, fixed with 15 mL saturated mercuric
chloride and then sealed with a rubber lid and aluminum cap
(Wheaton, USA). DIC samples were then run on a custom DIC
system with a LICOR gas analyzer (Rob Dunbar lab, Stanford,
USA) with a Dickson sea water reference run every 7 samples.
Carbon species concentration and aragonite state were determined
for each treatment using the CO2SYS program using the
dissociation constants from [72] and refit by [73] using TA,
DIC, pH, and temperature measurements [74].
Respirometry MeasurementsCoral branches designated for physiological measurements were
used for respirometry assays. The coral branches were dark
adapted for at least an hour before respirometry assays, which
were performed after dusk. Branches were placed in 70 cm3 clear
acrylic chambers with an inserted optode sensor connected to an
Oxy4 v2 system (PreSens, Regensburg, Germany). The chambers
were placed within an acrylic container (on top of a magnetic
stirrer), which was connected to a water bath keeping the
temperature constant throughout the assays at 26uC, the ambient
water temperature at Heron Island during the course of the
experiment. Chambers were filled with seawater from respective
experimental treatments in the aquaria. Photosynthesis vs
irradiance curves (P-E curves) were conducted by using the actinic
light source of an imaging pulse amplitude modulation fluorom-
eter (iPAM, Walz, Effeltrich, Germany), and exposing coral
branches in chambers to 0, 10, 20, 55, 110, 925 mmol quanta
m22 s21 for 10 min and then followed by exposure at 1075 and
1250 mmol quanta m22 s21 for 5 min each, and the run was
concluded by a 10 min incubation at 0 mmol quanta m22 s21.
Respirometry rates were normalized to cell number and coral
surface area. The exposure to different light levels enabled the
calculation of P-E curve parameters [75] following methods in
[15]; dark respiration (Rdark) estimated from the initial 10 min
dark incubation, sub-saturation photosynthetic efficiency (a)
derived from the regression line slope of the low irradiance levels
(10, 20,55, 110 mmol quanta m22 s21) in relation to the estimated
Ek, photosynthetic capacity (Pnet max) was estimated as the
greatest rate of oxygen evolution at the high irradiance levels (925,
1075, 1250 mmol quanta m22 s21), and finally light-enhanced
dark respiration (LEDR) was determined from the oxygen
consumption in the last dark incubation post irradiance exposure.
Population Density and Chlorophyll a Content ofSymbiodinium
The cell density and pigment content of Symbiodinium were
measured by removing tissue from coral fragments by air-
brushing frozen fragments in 5 mL 0.06 M phosphate buffer
(pH 6.65). The homogenate was centrifuged at 40006g for 5 min.
The supernatant was removed and the remaining dinoflagellate
pellet was re-suspended in filtered seawater (0.45 mm) and
separated into aliquots that were used for pigment quantification
and Symbiodinium cell counts. Symbiodinium pigment quantification
aliquots were centrifuged at 40006g for 5 min, the supernatant
was removed and 1 mL of 100% cold methanol was added to the
pellet. The solution was sonicated on ice cold water for 10 min
and then centrifuged at 40006g for 5 min. The supernatant was
collected and transferred into a tube. This process was repeated
until complete pigment extraction was achieved (when the final
supernatant was clear). The total final extracted solution was
filtered (0.45 mm) and used for pigment separation in a Shimadzu
SCL – 10 HPLC linked to a Shimadzu SPD – M10A photodiode
array detector, using the column and method described in [76]
with solutions A (methanol: acetonitrile: aquose pyridine, 50:25:25
v:v:v) and B1 (methanol: acetonitrile: acetone, 20:60:20 v:v:v). A
standard for methanol extracted pigment chlorophyll a was used
for quantifying pigments and normalized on a per cell basis.
Symbiodinium cell counts were estimated using eight randomly
selected replicates counted using a haemocytometer (Boeco,
Germany) on a Zeiss standard microscope; the counts were
normalized to coral surface area in cm2, as obtained by dipping
coral fragments into paraffin wax following the method of [77].
Coral Growth Rate EstimationAcropora millepora branch growth/calcification rate was estimated
using the buoyant weight method [78]. Coral branch weights for
samples across treatments were measured at time zero, day 1 and
day 28. The branches were suspended by a thin fishing line below
a precision balance (Mettler Toledo) and the weight was recorded.
The growth/calcification rate was calculated as a relative unit by
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subtracting the initial weight (g) from the final weight (g) and
converting this to a percent change in weight over the course of
the experimental period.
Statistical AnalysisAll data were tested for normality and homogeneity of variance
and where assumptions were violated, the data were corrected by
transformations. Non-parametric equivalents of tests were used in
cases where assumptions were violated despite transformations. A
Kruskal Wallis test was used to determine the effect of changes in
CO2 concentrations on Symbiodinium density, branch calcification/
growth, Pnet max, Pgross max, LEDR and Rdark. To test for
significance on the expression levels of mRNAs from quantitative
real time PCR between control and high CO2 levels, for each gene
a Welch t-test was used. All statistical analyses were performed
using STATISTICA 7.0 (Statsoft Inc., Tulsa, USA).
Microarray DescriptionThe microarrays used in this experiment were printed at the
Adelaide Microarray Facility (Australia) and consisted of 18,432
spots derived from the same amount of cDNA clones, including
290 spots representing positive and negative control and
representing 8606 unigene clusters [79]. These microarrays are
the 3rd generation cDNA microarrays designed for Acropora
millepora [80]. The selection of clones, methodological approach
for the cDNA library construction and the fabrication of
microarrays are explained in [81].
Hybridization of ArraysTotal RNA was extracted from each sample using Trizol
(Invitrogen) following manufacturer’s instructions. The integrity
and quality of total RNA was assessed using a Bioanalyzer (Agilent
Technology). Only samples showing intact RNA (RNA Integrity
number .8), were used for probe construction. cDNA probe
synthesis was performed from 1000 ng total RNA using
Superscript Reverse Transcriptase (Invitrogen) and a 2 pmol
Genisphere 900 3DNA Dendrimer from a Genisphere 3DNA-900
microarray kit according to the manufacturers’ instructions. We
used a reference two-colour microarray design, where, for each
array, the sample was labeled with Cy5 and the reference,
consisting of pooled RNA from control treatments and time zero,
was labeled with Cy3. In total, 27 arrays were hybridized, as each
array represented a sample from a treatment and a time point
(n = 3), only 3 out of the 4 colonies were used for microarray
hybridization. Microarrays were pre-hybridized and hybridized
with the labeled samples using the Genisphere 3DNA-900
microarray kit following the manufacturer’s instructions and using
a dynamic hybridization system (MAUI, BioMicro Systems). Prior
to and post hybridization, the microarray slides were washed three
times (wash 1: 26SSC 0.2% SDS at 65uC for 15 min, wash 2:
26SSC at room temperature for 10 min, wash 3: 0.26SSC at
room temperature for 10 min). Slides were scanned using a
GenePix H 4200 scanner (Axon Instruments) and image
acquisition was performed using the software GenePix H Pro 5
(Molecular Devices, CA, USA).
Microarray AnalysisNormalization and data analysis of acquired array slides was
performed using R (R Development Core Team, 2008) and the
limma package [82]. The details for the methodology of analyzing
differential gene expression using empirical Bayes shrinkage of
variance and linear regression models can be found in [83].
Normexp (75) corrected signal intensities were used, as it has been
shown to be a well performing background correction method,
which best stabilizes variance as a function of intensity, compared
to more standard and common methods [84]. Print-tip loess
normalization was applied within slides [85] while scale normal-
ization was applied between slides, in order to ensure that
distributions were similar between arrays. Both normalization
procedures equalize for differing amounts of host RNA input [36].
Effectiveness of normalization procedures was verified through M
(the log ratio of the spot fluorescence intensity) vs A (the log of the
average spot fluorescence intensity) plots. Minimal or no
fluorescence was observed for probes which contained salmon
sperm DNA and primers, and should not hybridize, while controls
which were expected to hybridize showed a range of fluorescence
intensities. Differentially expressed genes were identified based on
an assumed false discovery rate of 5% and sequence-wise p-values
were adjusted through the Benjamini and Hochberg method [86].
Sets of contrast lists of differentially expressed genes between
control, medium and high CO2 treatments at day 1 and day 28
were created (643 transcripts) (Table S1, Additional file 1), in
addition contrast lists were created between groups of samples at
t0 so that any potential differences due to ‘‘tank effects’’ could be
subtracted from subsequent analysis; in total 3 genes were
subtracted from subsequent contrast lists. Differentially expressed
genes at day 1 and 28 were then assembled into 6 different clusters
based on their temporal gene expression patterns, using K-means
clustering analysis in the TIGR TMEV software [87], assuming that
genes with similar cellular pathways share common temporal
expression patterns. In addition Principal component analysis was
also carried out in the TIGR TMEV software [87]. Differentially
expressed genes which had homology to known genes (352
transcripts, Blastx, E-score cutoff 1026), were assigned to GO
categories and subjected to classification analysis using the
hypogeometric test and a false discovery rate of 5% in GOEAST
[88] to identify enriched GO groups. Microarray data has been
deposited in the Gene Expression Omnibus Database (GSE28697).
Validation by Quantitative PCRExpression patterns of candidate genes from each functional
group of coral genes differentially expressed in response to
increased CO2 treatment (sodium and chloride transporter, V
type ATPase, vitellogenin, catalase, caspase 3, calmodulin,
cytoskeletal actin, pyruvate dehydrogenase, G protein and rab
protein) were validated through quantitative Polymerase Chain
Reaction (qPCR). Total RNA (1000 ng) was reverse transcribed
with a Superscript Vilo cDNA synthesis kit (Invitrogen) following
manufacturer’s instructions. Specific primers amplifying approx-
imately 100–200 bp PCR products were designed for the genes
(Table S4) chosen to be validated from the microarray data.
Transcript levels were determined by qPCR using the Corbett
Rotor Gene 6000 thermal cycling system (Qiagen), following the
manufacturer’s instructions (Qiagen, CA, USA) and PCR
conditions (95uC for 10 min, followed by 40 cycles of 95uC for
15 sec and 60uC for 1 min). Triplicate first strand diluted 1:10
cDNA aliquots (1 mL) from each sample were used in 20 mL PCR
reactions with 2 mM primers and a SYBR Green PCR master mix
(Warrington, Cheshire, UK). For each candidate gene control
versus high CO2 samples (4 replicates) at day 28 were tested, as
this was the conditions under which major physiological changes
occurred, and the greatest differential gene expression was present.
A no template control as well as a no reverse transcription control
was performed for each gene and treatment to ensure that the
cDNA samples did not have DNA contamination. In addition to
ensure that gene expression data was representing coral host
genes, primer specificity of all primers was tested through PCR
Cellular Impacts of Ocean Acidification
PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e34659
using the qPCR primers and cDNA and genomic DNA from
Symbiodinium sp. as a template to ensure no amplification. The
comparative delta CT method was used to determine relative
quantities of mRNA transcripts from each sample. Each value was
normalized to two reference genes adenosyl-homocysteinase
(AdoHcyase) and ribosomal protein L7 (Rpl7). The selection of
reference genes for this experiment was done by using a pool of
reference genes (Table S4) and analyzing the expression stability
using the GeNorm software [89]. For this study the most stable
expression was found for adenosyl-homocysteinase (AdoHcyase)
and ribosomal protein L7 (Rpl7) (M value = 0.253) and a
minimum of two reference genes was recommended (V2/
3 = 0.126). Relative expression values were for each gene were
calculated by showing a ratio of treatment relative expression over
control relative expression on a log2 scale, which provides a similar
appearance of up and down regulation [41].
Supporting Information
Figure S1 Principal component analysis of gene expres-sion for 3 acidification treatments; control (c), medium(m) and high (h) and 3 time points: T0 (t), day 1 (d) andday 28 (m).(EPS)
Table S1 Annotated list of differentially expressedtranscripts for Acropora millepora between CO2 at day1 and day 28 (from Additional file 1), as determined byempirical Bayes moderated statistics. Transcripts are
assembled into 6 different clusters according to K-means clustering
and based on their temporal gene expression patterns. Annotations
represent blastx, results with an E-score cutoff 1026.
(XLS)
Table S2 Summary of t-test with Welch’s correctionresults for candidate transcripts validating microarrayresults for control and high treatments at day 28 (n = 4).
(EPS)
Table S3 Comparison of mean log2 fold changes forcandidate transcripts from qPCR and microarray dataanalysis, at day 28 for high versus control corals. The
correlation coefficient (R) is 0.93.
(EPS)
Table S4 List of candidate genes used in qPCRexpression analyses.
(EPS)
Acknowledgments
We thank A. Forrest for critical discussions. Microarray data are Minimum
Information About a Microarray Experiment (MIAME) compliant and
deposited under accession number GSE28697 (National Center for
Biotechnology Information GEO).
Author Contributions
Conceived and designed the experiments: PK DK MRL OHG. Performed
the experiments: PK DK. Analyzed the data: PK PC SD. Contributed
reagents/materials/analysis tools: PK PC OHG DM. Wrote the paper: PK
PC SD OHG DK DM MRL.
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Cellular Impacts of Ocean Acidification
PLoS ONE | www.plosone.org 12 April 2012 | Volume 7 | Issue 4 | e34659